Immunomodulation by poly-YE reduces organophosphate-induced brain damage

Abstract

Accidental organophosphate poisoning resulting from environmental or occupational exposure, as well as the deliberate use of nerve agents on the battlefield or by terrorists, remain major threats for multi-casualty events, with no effective therapies yet available. Even transient exposure to organophosphorous compounds may lead to brain damage associated with microglial activation and to long-lasting neurological and psychological deficits. Regulation of the microglial response by adaptive immunity was previously shown to reduce the consequences of acute insult to the central nervous system (CNS). Here, we tested whether an immunization-based treatment that affects the properties of T regulatory cells (Tregs) can reduce brain damage following organophosphate intoxication, as a supplement to the standard antidotal protocol. Rats were intoxicated by acute exposure to the nerve agent soman, or the organophosphate pesticide, paraoxon, and after 24 h were treated with the immunomodulator, poly-YE. A single injection of poly-YE resulted in a significant increase in neuronal survival and tissue preservation. The beneficial effect of poly-YE treatment was associated with specific recruitment of CD4⁺ T cells into the brain, reduced microglial activation, and an increase in the levels of brain derived neurotrophic factor (BDNF) in the piriform cortex. These results suggest therapeutic intervention with poly-YE as an immunomodulatory supplementary approach against consequences of organophosphate-induced brain damage.

Introduction

Nerve agents (e.g., soman, tabun, sarin, cyclosarin, and VX) are organophosphorous compounds that act by inhibiting the enzyme acetylcholinesterase, and are considered to be the most toxic of all chemical weapons. Their neurotoxic mechanism is also shared by organophosphate pesticides, which are one of the most common products of the modern chemical industry. Large scale release of organophosphate pesticides to the environment and their high availability frequently lead to intoxications, which result in long term morbidity and even in death. The widespread use of these compounds and their high neurotoxicity, not only constitutes a hazard of everyday life, but also establishes organophosphate pesticides, along with nerve agents, as a potential weapon (Colosio et al., 2003, Hoffman et al., 2007, Okumura et al., 1996, Sidell et al., 2008). To date, no treatments aimed at counteracting secondary degeneration following organophosphate poisoning are available (Layish et al., 2005, Markel et al., 2008, Sidell et al., 2008). While having no direct effect on survival of the patients, such treatments might be particularly important for counteracting organophosphate-induced cognitive and psychopathological impairments (Jamal et al., 2002, Levin et al., 1976, Rosenstock et al., 1991, Stallones and Beseler, 2002).

Organophosphate-induced brain injury is known to activate microglia (Zimmer et al., 1997). For decades, microglial activation was considered to be destructive in the context of the central nervous system (CNS) tissue, contributing to the secondary degeneration following injury (Liu et al., 2002). However, it was subsequently demonstrated that if microglial cells are properly regulated by T cell-derived cytokines or by cytokines released from blood-derived macrophages, they can support buffering of excessive levels of glutamate, secrete neurotrophic factors, induce neurogenesis, and promote tissue repair (Beers et al., 2008, Butovsky et al., 2006, Chiu et al., 2008, Ekdahl et al., 2009, El Khoury et al., 2007, Shaked et al., 2004, Simard et al., 2006). Specifically, in acute CNS insult, termination of the microglial activity via the recruitment of interleukin (IL)-10-secreting monocytes induced by T-cell based immunization was shown to be beneficial for recovery (Shechter et al., 2009). Moreover, the spontaneous recovery from CNS insults and the ability to cope with mental stress was shown to be T cell-dependent (Cohen et al., 2006, Kipnis et al., 2004b, Lewitus and Schwartz, 2009, Moalem et al., 1999, Yoles et al., 2001). Since basal immune maintenance is not sufficient to counteract the forces that drive neurotoxicity, boosting the protective T-cell response can be accomplished either by active immunization with the relevant self CNS antigens or via modulation of the naturally occurring T regulatory cells (Tregs), which constitutively suppress potential anti-self helper (CD4⁺) T cells (Kipnis et al., 2002, Schwartz and Ziv, 2008). One way to achieve this aim is by administration of poly-YE, a high-molecular-weight copolymer (22–45 kDa) of glutamate and tyrosine with immunomodulatory properties (Cady et al., 2000, Vidovic and Matzinger, 1988), which is able to down-regulate the suppressive properties of Tregs (Ziv et al., 2007). This prompted us to test the neuroprotective potential of poly-YE in a model of soman-induced brain damage, and to further characterize the neuroimmune effects of poly-YE in a model of intoxication with paraoxon, a highly toxic metabolite of the organophosphate pesticide, parathion.